A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein-Protein Interactions.,ACS Chemical Biology

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A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein-Protein Interactions.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-12-06 , DOI: 10.1021/acschembio.9b00669
Julia Zinkus-Boltz ₁ , Craig DeValk ₂ , Bryan C Dickinson ₁

Affiliation

Protein-protein interactions (PPIs) are critical for organizing molecules in a cell and mediating signaling pathways. Dysregulation of PPIs is often a key driver of disease. To better understand the biophysical basis of such disease processes-and to potentially target them-it is critical to understand the molecular determinants of PPIs. Deep mutational scanning (DMS) facilitates the acquisition of large amounts of biochemical data by coupling selection with high throughput sequencing (HTS). The challenging and labor-intensive design and optimization of a relevant selection platform for DMS, however, limits the use of powerful directed evolution and selection approaches. To address this limitation, we designed a versatile new phage-assisted continuous selection (PACS) system using our previously reported proximity-dependent split RNA polymerase (RNAP) biosensors, with the aim of greatly simplifying and streamlining the design of a new selection platform for PPIs. After characterization and validation using the model KRAS/RAF PPI, we generated a library of RAF variants and subjected them to PACS and DMS. Our HTS data revealed positions along the binding interface that are both tolerant and intolerant to mutations, as well as which substitutions are tolerated at each position. Critically, the "functional scores" obtained from enrichment data through continuous selection for individual variants correlated with KD values measured in vitro, indicating that biochemical data can be extrapolated from sequencing using our new system. Due to the plug and play nature of RNAP biosensors, this method can likely be extended to a variety of other PPIs. More broadly, this, and other methods under development support the continued development of evolutionary and high-throughput approaches to address biochemical problems, moving toward a more comprehensive understanding of sequence-function relationships in proteins.

中文翻译：

蛋白质-蛋白质相互作用的深突变扫描的噬菌体辅助连续选择方法。

蛋白质-蛋白质相互作用（PPI）对于组织细胞中的分子和介导信号通路至关重要。PPI的失调通常是疾病的主要驱动因素。为了更好地了解此类疾病过程的生物物理基础，并潜在地针对它们，了解PPI的分子决定因素至关重要。深度突变扫描（DMS）通过将选择与高通量测序（HTS）耦合来促进大量生化数据的采集。然而，DMS相关选择平台的艰巨且费力的设计和优化限制了强大的定向进化和选择方法的使用。为了解决这个限制，我们使用先前报道的邻近依赖性分裂式RNA聚合酶（RNAP）生物传感器设计了一种多功能的新型噬菌体辅助连续选择（PACS）系统，目的是大大简化和简化PPI的新选择平台的设计。在使用模型KRAS / RAF PPI进行表征和验证后，我们生成了一个RAF变体库，并对其进行了PACS和DMS处理。我们的HTS数据揭示了沿着结合界面的位置既可以耐受突变，也可以耐受突变，并且每个位置都可以耐受取代。至关重要的是，通过连续选择单个变体从富集数据中获得的“功能评分”与体外测得的KD值相关，这表明可以使用我们的新系统从测序中推断出生化数据。由于RNAP生物传感器的即插即用特性，该方法可能会扩展到多种其他PPI。更广泛地说，该方法和其他正在开发的方法支持不断发展的进化和高通量方法来解决生物化学问题，从而使人们对蛋白质的序列-功能关系有了更全面的了解。

更新日期：2019-12-07

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